Novel levamisole derivative induces extrinsic pathway of apoptosis in cancer cells and inhibits tumor progression in mice

Novel Levamisole Derivative Induces Extrinsic Pathwayof Apoptosis in Cancer Cells and Inhibits TumorProgression in MiceMahesh Hegde1, Subhas S. Karki2, Elizabeth Thomas1, Sujeet Kumar2, Kuppusamy Panjamurthy1,

Somasagara R. Ranganatha1, Kanchugarakoppal S. Rangappa3, Bibha Choudhary4,

Sathees C. Raghavan1*

1 Department of Biochemistry, Indian Institute of Science, Bangalore, Karnataka, India, 2 Department of Pharmaceutical Chemistry, KLE University’s College of Pharmacy,

Bangalore, Karnataka, India, 3 Department of Studies in Chemistry, University of Mysore, Mysore, Karnataka, India, 4 Institute of Bioinformatics and Applied Biotechnology

(IBAB), Bangalore, Karnataka, India

Abstract

Background: Levamisole, an imidazo(2,1-b)thiazole derivative, has been reported to be a potential antitumor agent. In thepresent study, we have investigated the mechanism of action of one of the recently identified analogues, 4a (2-benzyl-6-(49-fluorophenyl)-5-thiocyanato-imidazo[2,1-b][1,3,4]thiadiazole).

Materials and Methods: ROS production and expression of various apoptotic proteins were measured following 4atreatment in leukemia cell lines. Tumor animal models were used to evaluate the effect of 4a in comparison with Levamisoleon progression of breast adenocarcinoma and survival. Immunohistochemistry and western blotting studies wereperformed to understand the mechanism of 4a action both ex vivo and in vivo.

Results: We have determined the IC50 value of 4a in many leukemic and breast cancer cell lines and found CEM cells mostsensitive (IC50 5 mM). Results showed that 4a treatment leads to the accumulation of ROS. Western blot analysis showedupregulation of pro-apoptotic proteins t-BID and BAX, upon treatment with 4a. Besides, dose-dependent activation of p53along with FAS, FAS-L, and cleavage of CASPASE-8 suggest that it induces death receptor mediated apoptotic pathway inCEM cells. More importantly, we observed a reduction in tumor growth and significant increase in survival upon oraladministration of 4a (20 mg/kg, six doses) in mice. In comparison, 4a was found to be more potent than its parentalanalogue Levamisole based on both ex vivo and in vivo studies. Further, immunohistochemistry and western blottingstudies indicate that 4a treatment led to abrogation of tumor cell proliferation and activation of apoptosis by the extrinsicpathway even in animal models.

Conclusion: Thus, our results suggest that 4a could be used as a potent chemotherapeutic agent.

Citation: Hegde M, Karki SS, Thomas E, Kumar S, Panjamurthy K, et al. (2012) Novel Levamisole Derivative Induces Extrinsic Pathway of Apoptosis in Cancer Cellsand Inhibits Tumor Progression in Mice. PLoS ONE 7(9): e43632. doi:10.1371/journal.pone.0043632

Editor: Rafael Moreno-Sanchez, Instituto Nacional de Cardiologia, Mexico

Received February 13, 2012; Accepted July 23, 2012; Published September 10, 2012

Copyright: � 2012 Hegde et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was supported by grant from Lady Tata Memorial Trust, United Kingdom, and Indian Institute of Science start up grant for SCR. The funderhad no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Competing Interests: The authors have declared that there is no competing interest.

* E-mail: [email protected]

Introduction

Cancer is a difficult disease to treat, and only very few effective

drugs are available. The development of novel, efficient, selective

and less toxic cancer therapeutic molecules has been a challenging

goal. Understanding the molecular mechanism involved in cancers

will lead to the discovery of novel anticancer agents. Changes in

expression levels of RNA and proteins due to different mutations

have been studied in many cancers, including leukemia and

lymphoma [1–4]. Recently, there have been extensive efforts to

characterize the mechanism of chromosomal translocations and

deletions resulting in leukemia and lymphoma [5,6]. Many gene

fusions have also been identified in prostate cancers and breast

cancers [7]. The most discussed proteins responsible for leukemia

and lymphoma in the recent past are the recombination activating

genes (RAGs, the enzyme responsible for antibody diversity) [5,6]

and activation induced deaminase (AID, the enzyme responsible

for somatic hypermutation and class switch recombination) [5,8].

However, the enzymes responsible for the development of gene

fusions are yet to be identified.

The past two decades have seen a dramatic change in cancer

treatment paradigms. For example, Imatinib (Gleevac), a drug

developed specifically against the activated tyrosine kinase in

chronic myelogenous leukemia, is one of such major advances [9].

In addition, many other compounds have also been identified and

clinically tested. Although, the success of clinical trials in

identifying new agents and treatment modalities has been

significant, the current treatments have many limitations. This

PLOS ONE | www.plosone.org 1 September 2012 | Volume 7 | Issue 9 | e43632

includes side effects induced by the drugs and acquired drug

resistance [10]. Thus, the need for the development of effective

anti-cancer therapeutic agents with well-defined pharmacokinetic

properties is of great importance.

Currently, there are different ways by which a drug is tested for

its effectiveness as an anticancer agent. In this regard, various

apoptotic pathways have been studied extensively for many

compounds to understand their mode of cytotoxicity [11]. Cell

cycle check points induced by small molecules have also been

investigated [12,13].

Levamisole is an immunomodulator in different cancer cells

including colorectal, breast cancer, melanoma, and leukemia [14].

Previously, it has been shown that it affects cell proliferation in

different cancers [15] and modulates the phosphorylation relevant

for both cell cycle progression and apoptosis. Studies have also

shown that it can be used for anti- helminthic infestations and

various autoimmune diseases [16,17]. Besides, it has been shown

that levamisole has anticancer activity in combination with

fluorouracil (5-FU) as adjuvant therapy for tumor-node-metastasis

(TNM) stage III (Dukes’ C) colon carcinoma [18].

The imidazo(2,1-b)thiazole derivatives of Levamisole have been

reported as potential antitumor agents [19]. Later, antitumor

activity of 5-formyl-6-arylimidazo-[2,1-b]-1,3,4-thiadiazole sulfon-

amides were also reported [20]. Based on these promising results,

we synthesized a series of analogues containing fluorine at position

4 of 6-phenyl in imidazo-[2,1-b]-1,3,4-thiadiazole and identified

4a as the lead compound [21]. However, the mechanism by which

it induced cytotoxicity was not known. Besides, it was never tested

on animal models for its effect on tumor progression. In the

present study, we report that 4a exerts its effect on tumor cells by

activating the extrinsic pathway of apoptosis. We also found that

4a inhibits the progression of tumor in mice effectively and

increases the lifespan significantly.

Materials and Methods

Chemicals and reagentsAll the chemicals used in the present study were of analytical

grade and purchased from Sigma–Aldrich, USA. Antibodies were

obtained from Santa Cruz Biotechnology, USA.

Synthesis of 4aSynthesis and characterization of 2-benzyl-6-(49-fluorophenyl)-

5-thiocyanato-imidazo[2,1-b][1,3,4]thiadiazole, 4a has been de-

scribed earlier [21]. Levamisole (Tetramisole hydrochloride, Cat.

No. L9756) was purchased from Sigma-Aldrich, USA.

Cell cultureHuman cell lines, CEM (T-cell leukemia), K562 (Chronic

myelogenous leukemia) REH (B-cell leukemia) and Nalm6 (B-cell

leukemia), were cultured in RPMI1640 (Sera Lab, UK) containing

10% FBS (Gibco BRL, USA), 100 U of Penicillin G/ml and

100 mg of streptomycin/ml (Sigma–Aldrich, USA) at 37uC in a

humidified atmosphere containing 5% CO2. EAC (breast cancer)

cell line was purchased from National Center for Cell science,

Pune and grown in DMEM containing 10% FBS as described

above.

Trypan blue dye exclusion assayThe effect of 4a on viability of leukemic (CEM, K562, REH,

Nalm6) and breast cancer (EAC) cells were determined by Trypan

blue dye exclusion assay [22]. Cells were cultured (0.756105 cells/

ml) for 24 h and compound was added in the range of 1–100 mM

to determine the IC50 value. DMSO treated cells were used as

vehicle control. Cells were collected at intervals of 24 h for five

days and number of viable cells was determined following trypan

blue staining. For Levamisole, water was used as vehicle control.

In case of EAC, an adherent cell line, viability was measured at 48

and 72 h after treatment of 4a. Each experiment was repeated a

minimum of two times and error bars were calculated and plotted.

MTT assayThe MTT assay was performed as described earlier [23]. CEM,

K562, REH or Nalm6 cells (0.756105 cells/ml) were treated with

4a (for CEM and REH cells 1, 5, 10 and 20 mM; for K562 1, 5,

10, 20, 40 and 100 mM; for Nalm6, 1,5,10, 20, 40 mM), incubated

for 48 and 72 h and subjected to MTT assay. Cells treated with

DMSO or water was used as vehicle controls for 4a, respectively.

Experiment was repeated a minimum of two independent times,

each with duplicate reactions and the error bars are indicated.

LDH release assayLDH release into media following 4a treatment (1, 5, 10 and

20 mM) on CEM cells after 48 and 72 h of treatment was

measured using standard protocol [24]. The percentage of LDH

release was calculated as: LDH release in media/(LDH release in

media+intracellular LDH release)6100%.

Detection of intracellular ROS production by flowcytometry

The level of total intracellular ROS production was measured

by using cell permeable fluorescent probe 2,7-dichlorodihydro

fluorescein diacetate (H2DCFDA) in CEM and REH cells [25].

CEM cells were treated with 5 and 10 mM of 4a and REH cells

with 10 mM for 5, 10, 15, 30 and 60 min, harvested, washed and

the fluorescence intensity was analyzed by flow cytometry. Cells

treated with H2O2 were used as positive control for compensation

of experimental samples.

Western blot analysisCell lysate was prepared following treatment with 4a on CEM

(0, 0.5, 1, and 5 mM for 48 h). Western blotting was performed as

described previously [23]. Briefly, ,40 mg of protein sample was

electrophoresed on 8–12% SDS-PAGE, transferred to PVDF

membrane (Millipore, USA) and probed with respective primary

and biotinylated secondary antibodies. The primary antibodies

used were BCL2, BCL-xL, BAX, t-BID, p53, p-p53 [Ser 392],

PUMA, AKT, pAKT [Ser 473], FAS, FAS-L, FADD, SMAC/

DIABLO, CASPASE-3, CASPASE-8 and CYTOCHROME C.

The blots were developed using chemiluminescent reagents

(ImmobilonTM western, Millipore, India) and scanned by gel

documentation system (LAS 3000, Fuji, Japan). Blots were

stripped subsequently as per standard protocol and re-probed

with anti-TUBULIN antibody [23].

Separation of mitochondrial and cytosolic fractions from4a treated CEM cells

CEM cells were treated with 5 mM 4a for 48 h, harvested and

used for isolation of mitochondrial and cytosolic fractions using

mitochondrial extraction kit (IMGENEX, USA, Cat. No. 10082k)

as per the manufacturer’s instructions. DMSO treated cells were

used as control. The resulting fractions were used for western blot

analysis against anti-CYTOCHROME C. Actin was used as

loading control.

Levamisole Derivative: Novel Anti-Cancer Agent

PLOS ONE | www.plosone.org 2 September 2012 | Volume 7 | Issue 9 | e43632

In vivo experimentsEthics Statement. Mice were maintained as per the princi-

ples and guidelines of the ethical committee for animal care of

Indian Institute of Science in accordance with Indian National

Law on animal care and use. The experimental design of the

present study was approved by Institutional Animal Ethics

Committee (Ref. CAF/Ethics/125/2007/560), Indian Institute

of Science, Bangalore, India.

AnimalsSwiss albino mice, 6–8 weeks old, weighing 18–22 g were

purchased from central animal facility, Indian Institute of Science

(IISc), Bangalore, India and maintained in the animal house,

Department of Biochemistry, IISc. The animals were housed in

polypropylene cages and provided standard pellet diet (Agro

Corporation Pvt. Ltd., India) and water ad libitum. The standard

pellet diet composed of 21% protein, 5% lipids, 4% crude fiber,

8% ash, 1% calcium, 0.6% phosphorus, 3.4% glucose, 2%

vitamin, and 55% nitrogen-free extract (carbohydrates). The mice

were maintained under controlled conditions of temperature and

humidity with a 12 h light/dark cycle.

Preparation of Ehrlich ascites carcinoma (EAC)

cells. EAC cells were collected from the peritoneal cavity of

tumor-bearing donor mice of 20–22 g body weight and suspended

in sterile phosphate buffered saline (PBS). A fixed number of viable

cells (16106 cells/22 g b. wt) were implanted into the peritoneal

cavity of each recipient mouse and allowed to multiply. The tumor

cells were withdrawn, diluted in saline, counted and re-injected

(16106 cells/animal) to right thigh tissue of experimental animals

for developing solid tumor.

Evaluation of antitumor activity of 4a in mouseTo study and compare the antitumor activity of 4a, 32 Swiss

albino mice were used in the present study, (two batches of 16

animals each). Out of 16 mice, four were used as untreated

(normal) control. Rest of the mice were injected with EAC to

induce solid tumor, and divided into three groups, each containing

four animals for tumor control (group two), Levamisole treated

(group three) and 4a treated (group four). Group two received

water as vehicle control, group three received oral administration

of Levamisole (20 mg/kg, b. wt) and group four received oral

administration of 4a (6 doses of 20 mg/kg) on every alternative

day using gastric gavages starting from 12th day of injection of

tumor cells.

The diameters of developing tumor were measured in the case

of group two, three and four animals by using vernier calipers once

in five days. Tumor volume was calculated using the formula

V = 0.5ab2, where ‘a’ and ‘b’ indicate the major and minor

diameter, respectively [26]. At the end of 25th and 45th day of

experimental period, one animal from each group was sacrificed

by cervical dislocation and tissues from normal (group one), tumor

(group two), Levamisole treated (group three) and 4a treated

(group four) animals were collected and stored.

To check the longevity induced by 4a in tumor mice, 24

animals were studied, two batches containing 12 each. Out of 12,

six served as tumor control and others were treated with 4a as

explained earlier. The percentage of increase in lifespan was

calculated and compared with that of control animals. The death

pattern for controls and 4a treated animals was recorded and %

increase in lifespan was calculated using the formula [(T2C)/

C]6100, where ‘T’ indicates the number of days the 4a treated

animals survived and ‘C’ indicates the number of days tumor

animals survived [26–28].

Evaluation of toxicity of 4a in normal miceSwiss Albino mice were treated with 4a and Levamisole (6

doses, on every alternate day) and side effects were evaluated at

two different time points (20th and 50th day). Out of 36 mice, 18

each were used at 20th and 50th day. In both cases, 6 animals

served as control, while 6 were treated with Levamisole (20 mg/

kg) or 4a (20 mg/kg). Body weight of each animal was monitored

throughout the experiment and average weight calculated at 20th

and 50th day for control, 4a and Levamisole administered mice

and were plotted with error bars. In order to evaluate the effect of

4a and Levamisole on physiological functions, blood was collected

on 20th and 50th day as described earlier [29]. Serum was

separated and liver and kidney function tests were performed for

each animal, to determine levels of alkaline phosphatase (ALP),

creatinine, urea and blood count was carried out using plasma as

described earlier [29]. Values are presented as mean6SEM.

Western blot analysis for 4a treated solid and liquidtumor cells

Solid tumor was developed as described previously [29].

Following 6 doses of 4a, tumor was collected and extract was

prepared using RIPA buffer method [23]. The liquid tumor was

developed by injecting EAC cells (26106 cells) from donor mice to

peritoneal cavity of the experimental animals. Following EAC

injection (5th day), animals were treated with 4a (20 mg/kg; 4

doses every alternative days) and EAC cells were isolated from the

peritoneal cavity. The macrophage lineage cells were separated

from non-adherent EAC cells by gentle aspiration, and washed

with 16 PBS. Cell viability was checked using trypan blue dye

exclusion assay and 92% cells were found to be alive in both

experimental and controls groups. EAC cells were lysed in RIPA

buffer, extract was prepared as described earlier [30] and used for

western blot analysis.

Histological evaluationTumor and liver tissues of normal and experimental mice were

collected and processed as per standard protocols. Briefly, the

tissues were embedded in paraffin wax, sectioned at 5–10 mm in a

rotary microtome (Leica Biosystems, Germany) and stained with

haematoxylin and eosin [31,32]. Brain tissues were collected,

processed and stained with Luxol Fast Blue to study demyelin-

ation. Each section was evaluated by light microscopy and images

were captured (Zeiss, Germany).

Immunohistochemical (IHC) analysisAntibody staining was conducted on formalin fixed, paraffin

embedded tissues, which were sectioned at a thickness of 5 mm.

Slides were de-paraffinized using xylene, rehydrated and treated

with 3% H2O2 in PBS. Antigen retrieval was done using 0.01%

sodium-citrate buffer followed by blocking in PBST containing

0.1% BSA and 10% FBS. Primary antibody incubation (Ki67,

BID or 53BP1) was carried out overnight at 4uC. Slides were

washed and incubated with biotinylated secondary antibody (1 h).

Slides were then washed, incubated in streptavidin-HRP (1:1000).

Slides were again washed (PBS containing 0.1% Tween 20) and

colour was developed using DAB+H2O2, counterstained with

haematoxylin and mounted in DPX (Sigma-Aldrich, USA).

Images were captured using light microscope (Zeiss, Germany).

Change in intensity of antibody staining following 4a treatment

was determined by using ImageJ software [33].

Levamisole Derivative: Novel Anti-Cancer Agent

PLOS ONE | www.plosone.org 3 September 2012 | Volume 7 | Issue 9 | e43632

Statistical analysisValues are expressed as mean 6 SEM for control and

experimental samples and statistical analysis was performed using

One-way ANOVA followed by Dunnett test and each value was

compared with the control and significance is mentioned. For this

analysis, GraphPad software prism 5.1 was used. The values were

considered as statistically significant, if the p-value was equal to or

less than 0.05.

Results

4a induces cytotoxicity in cancer cellsPreviously, while screening a series of Levamisole derivatives,

we identified 4a as the lead compound (Fig. 1A) [21]. In the

present study, we have used a variety of leukemic cell lines (CEM,

K562, REH and Nalm6) and a mice breast cancer cell line,

Ehrlich ascites carcinoma (EAC) to evaluate its potential to induce

cytotoxicity. Firstly, IC50 of 4a on CEM, K562, Nalm6 and REH

cells was determined using trypan blue and MTT assays (Fig. 1).

Cells treated with DMSO were used as vehicle control. Results

showed that 4a treatment significantly affected cell viability at

lower concentrations in CEM, Nalm6 and REH (Fig. 1B, C).

Interestingly, K562 cells showed least sensitivity towards 4atreatment (Fig. 1B, C). Based on both trypan blue and MTT

assays, the IC50 value was estimated to be approximately 5, 70, 10

and 8 mM in CEM, K562, Nalm6 and REH cells, respectively

after 48 h of 4a treatment. Interestingly, in comparison with 4a,

Levamisole treatment on CEM cells showed less sensitivity

(Fig. 2A, B). 4a exhibited significant cytotoxicity in EAC cells

(IC50, 33 mM), while cells were insensitive to Levamisole, at the

range of concentrations tested (Fig. 2C, D). Further, LDH assay

was performed to assay cell damage induced by 4a on CEM cells.

Cells were treated with 4a for 48 and 72 h, respectively, harvested

and subjected to LDH measurement. Results showed a dose-

dependent increase in the release of LDH (Fig. S1).

4a induces intracellular reactive oxygen species (ROS)Overproduction of ROS following addition of a compound is an

indicator of cellular response leading to DNA damage and

apoptosis. We found that 4a treatment induced ROS production

in case of CEM (5 and 10 mM) as well as REH (10 mM) cells at 10

and 15 min (Fig. 3 A,B, Fig. S2). Further, the increase in

incubation time did not enhance the ROS level. Cells treated with

H2O2 were used as a positive control, while DMSO treated cells

served as vehicle control (Fig. 3). Thus, our results suggest that

ROS production is an intermediate step involved in 4a induced

cytotoxicity.

4a modulates expression of apoptotic proteinsIn order to study the mechanism by which 4a induces cell

death, we studied the expression levels of different apoptotic

proteins following 4a treatment. CEM cells were chosen for the

study as it showed the maximum sensitivity to 4a. CEM cells were

treated with increasing concentrations of 4a (0.5, 1 and 5 mM, for

48 h), cell lysate was prepared and used for western blot studies.

Results showed that 4a treatment led to a remarkable increase in

the levels of p53 as well as phospho-p53 (Fig. 4A). Since p53 is a

known activator of apoptosis, we tested the expression of various

BCL2 family proteins which have pro/antiapoptotic functions

(Fig. 4A, B). Consistent with our above results, we observed an

increase in the expression of proapoptotic proteins PUMA, BAX,

and cleavage of BID (Fig. 4A, B). Interestingly, we also observed

the upregulation of antiapoptotic proteins, BCL2 and BCL-xL,

particularly at 0.5 and 1 mM concentrations (Fig. 4B). Further, 4a

treatment led to the increase in expression of death-receptor

signaling proteins, FAS, FAS-L and FADD, indicating that

cytotoxicity induced by 4a could be mediated through the death

receptor mediated apoptosis (Fig. 4C).

PI3K/AKT pathway is known to be activated in a majority of

T-ALL. It is also known that it plays a critical role in controlling

survival and apoptosis. Increase in p-AKT shifts the cells towards

survival by interfering with p53 mediated pathway of apoptosis.

Hence, we were interested in checking the levels of AKT after

compound treatment. Results showed upregulation of AKT

following addition of 4a (Fig. 4A). Inspite of increase in the levels

of p-AKT, the drug induced cell death, which suggests that the

ratio of proapoptotic and antiapoptotic signals in the cell was

disrupted.

SMAC/DIABLO is a mammalian mitochondrial protein that

functions as a regulatory component during apoptosis. We tested

its expression upon 4a treatment and results showed an

upregulation of the protein expression (Fig. 4C). A dose-dependent

increase in the level of CYTOCHROME C was also observed

(Fig. 4D) [34]. We also checked for the release of CYTO-

CHROME C to cytoplasm and results showed a distinct increase

in the level of the cytosolic CYTOCHROME C upon treatment

with 4a (Fig. 4E).

CASPASE-8 is another protein activated during the extrinsic

pathway of apoptosis. Results showed cleavage of CASPASE-8

upon 4a treatment (Fig. 4D). This further confirms the activation

of the death-receptor mediated apoptosis. Activated CASPASE-8

also cleaves PROCASPASE-3 and consistent with this we find

activation of PROCASPASE-3 compared to the controls, upon 4atreatment, in a dose-dependent manner (Fig. 4D).

4a treatment inhibits tumor progression in miceEAC derived from breast adenocarcinoma is an aggressive and

rapidly growing carcinoma commonly used for the evaluation of

the effect of novel small molecules on tumor progression. Based on

pilot studies, 20 mg/kg body weight of 4a was used for treatment

in animals bearing tumors (data not shown). After 12th day of EAC

injection (small size tumor was visible), the animals were treated

with six doses every alternate day. We found that treatment with

4a on animals bearing tumor resulted in significant reduction of

tumor size compared to that of untreated as well as Levamisole

treated tumor animals (20 mg/kg) (Fig. 5A). We found that 80% of

the mice survived upon treatment with 4a, whereas 50% of the

untreated tumor mice were dead between 30 to 40 days of tumor

development (Fig. 5A, B and data not shown). The gross

appearance of thigh tissue containing tumor, liver and spleen of

negative control, untreated tumor control and 4a treated mice

showed a proportional morphological difference (Fig. 5C and data

not shown).

More importantly, we found that upon treatment with 4a,

animals with tumor showed a significant difference in the survival

rate compared to the untreated tumor control (Fig. 5B). While

control animals survived for only a maximum of 70 days after

tumor development, majority of mice treated with 4a survived for

more than 250 days indicating a ,4-fold increase in life span

(Fig. 5B). Therefore, our results demonstrate that 4a treatment

significantly reduced the tumor load and increased the lifespan of

the animals.

Histological evaluation was also performed at two different time

points (25th and 45th day) of treatment. Sections from tumor tissue

of a 25 day treated mouse showed many haematoxylin stained

nuclei with little cytoplasmic staining indicating active cell

proliferation, while in the case of controls, no other cells other

than the nuclei of skeletal muscles were stained (Fig. S3A). 4a

Levamisole Derivative: Novel Anti-Cancer Agent

PLOS ONE | www.plosone.org 4 September 2012 | Volume 7 | Issue 9 | e43632

treated tumor tissues showed a significant reduction in prolifer-

ating cells (Fig. S3A). Tissue sections from thigh after 45th day of

treatment showed negligible number of proliferating cells and were

more comparable with that of normal tissues, while proliferating

cells were abundant in mice bearing tumor, where no treatment

was given (Fig. S3A). To analyze whether 4a treatment had any

adverse effect on other tissues, sections of liver were analyzed by

haematoxylin and eosin staining (Fig. S3C,D). Our results showed

hypertrophy of hepatocytes in both tumor bearing and 4a treated

mice. However, it was restored back to normal only in cases where

the tumour regressed after treatment with compound 4a, unlike

the untreated mice where irregular hepatocytes were still seen (Fig.

S3C, D). Thus, our results show that 4a could be used as a potent

anticarcinogenic agent.

The effect of 4a on normal miceIt was important to study the side effects of 4a, as its parental

analogue Levamisole showed variety of side effects in animals as

well as human beings [35,36]. To assess the side effects of 4a and

Levamisole, it was orally administered to normal mice as described

in methods. Results showed significant increase in alkaline

phosphatase (ALP) level in case of Levamisole treated mice

(,50% increase compared to control) after 20 days of treatment.

Unlike, Levamisole, 4a showed only ,20% increase in ALP levels

(Fig. 6A). The liver sections also showed a similar effect (Fig. S4A).

Besides, kidney function tests for creatinine, urea also showed

comparable levels as in controls upon 4a treatment. WBC, RBC

counts and body weight were also found to be normal compared to

control in 4a and Levamisole treated cases (Fig. 6A, B). Brain

tissues were subjected to Luxol Fast Blue staining to check the

status of myelination. Results suggested that both the molecules

were nontoxic to the brain at the used concentration and doses.

Interestingly, 50th day post treatment showed normal ALP level in

serum in both the cases suggesting that local toxicity in liver

showed by both the molecules were transient and could be

recovered with time (Fig. 6C, D).

Treatment with 4a leads to reduction in proliferating cellswhile expression of apoptotic proteins increases in tumortissues

The Ki67 protein is expressed in all phases of the cell cycle

except G0 and is considered as a marker for cellular proliferation

[37,38]. The tumor cell proliferation was investigated by

immunohistochemical staining for Ki67 on tissue sections derived

from untreated and 4a treated tumors. Results showed efficient

Ki67 and nuclear staining in tumor sections, while the number of

Ki67 positive cells was substantially less in 4a treated tumors

(Fig. 7A, B). Further, we observed that the expression of p53

binding protein 1 (53BP1), and proapoptotic protein, BID was

significantly high following treatment with 4a in tumor tissues

(25th day of treatment) as compared to untreated tumor tissue

(Fig. 7C–F), further suggesting the activation of apoptosis in tumor

cells in mice. Therefore, our results show that 4a treatment

significantly inhibits tumor progression in mice.

Further, western blotting analysis was carried out on 4a treated

tumor cells from mice (both solid and liquid tumor) to evaluate the

effect of 4a on tumor progression (Fig. 8A, B). Results showed

Figure 1. Dose-dependent cytotoxic effect of 4a on leukemic cell lines. A. The structure of 4a. B. 4a induced cytotoxicity as determined bytrypan blue assay. CEM, K562, Nalm6 and REH cells were cultured (0.756105 cells/ml) and cytotoxicity was measured after addition of increasingconcentration of 4a as indicated. Cells were counted at intervals of 24 h until cells attained stationary phase and were plotted. DMSO treated cellswere used as vehicle control. Standard error was calculated based on minimum of two independent experiments. C. Determination of cellproliferation using MTT assay following addition of 4a to CEM, K562, Nalm6, and REH cells (48 and 72 h). Results shown are from a minimum of twoindependent experiments, each was done in duplicates and results are expressed as % of cell proliferation. In all panels ‘‘C’’ stands for DMSO treatedvehicle control.doi:10.1371/journal.pone.0043632.g001

Levamisole Derivative: Novel Anti-Cancer Agent

PLOS ONE | www.plosone.org 5 September 2012 | Volume 7 | Issue 9 | e43632

Figure 2. Comparison of cytotoxicity of 4a and Levamisole in CEM and EAC cells. A. The structure of Levamisole, the parental compoundof 4a. B. Determination of cell proliferation using MTT assay on CEM cells treated with Levamisole or 4a. In case of Levamisole, concentrations usedwere 1, 5, 10 and 20 mM, while it was 10 mM for 4a. Standard error was calculated based on two independent experiments. C, D. Cytotoxicity of 4aand Levamisole on EAC cells as measured by trypan blue assay. EAC cells were cultured (0.756105 cells/ml) and treated with 1, 5, 10, 20 and 40 mM of4a or Levamisole. Viability of the cells were determined by trypan blue assay at 48 and 72 h. Standard error was calculated based on threeindependent experiments.doi:10.1371/journal.pone.0043632.g002

Figure 3. Determination of intracellular ROS production in CEM and REH cells following treatment with 4a. A, B. CEM (A) and REH (B)cells treated with 4a (5 mM and 10 mM, respectively) for different time points were used for testing the formation of intracellular ROS by flowcytometry analysis. The concentration selected for the study was based on their respective IC50 values. H2O2 treated cells were used as positivecontrol while cells alone were used as negative control. DMSO treated cells were used as vehicle control. Cell population showing ROS was shownalong with standard error mean (n = 2).doi:10.1371/journal.pone.0043632.g003

Levamisole Derivative: Novel Anti-Cancer Agent

PLOS ONE | www.plosone.org 6 September 2012 | Volume 7 | Issue 9 | e43632

Figure 4. Expression of apoptotic proteins in CEM cells after 4a treatment. CEM cell lysate was prepared following treatment with 4a (0, 0.5,1 and 5 mM for 48 h). DMSO treated cells were used as control (0 mM). Western blotting studies were performed using specific primary and secondaryantibodies for expression of (A) Phospho p53, p53, PUMA, phospho AKT, AKT (B) BCL2, BCL-xL, BAX and t-BID; (C) FAS, FAS-L, FADD, and SMAC/DIABLO (D) CASPASE-3, CASPASE-8 and CYTOCHROME C. a-TUBULIN was used as loading control. The quantification of the bands in each blot shownin left panel is shown as bar diagram with standard error based on two independent experiments following normalization with respective TUBULIN E.Release of CYTOCHROME C from mitochondria after treatment with 4a. Mitochondrial as well as cytosolic fractions were separated from CEM cells

Levamisole Derivative: Novel Anti-Cancer Agent

PLOS ONE | www.plosone.org 7 September 2012 | Volume 7 | Issue 9 | e43632

upregulation of proapoptotic proteins, BAD and BAX in both

tumor models (Fig. 8A, B). We noted an upregulation of

expression of BCL2, which needs to be studied further. A

moderate downregulation of PCNA, a cell proliferation marker

was also observed, which is consistent with immunohistochemistry

results. Besides, we have also observed upregulation of both

activated and normal p53, FAS, FAS-L, FADD and CYTO-

CHROME C (Fig. 8A, B) suggesting that the mechanism of cell

death induced by 4a in tumor tissues within the animals and

cancer cell lines was comparable. We also observed cleavage of

CASPASE-8 in both cases although CASPASE-3 cleavage was

undetectable.

Discussion

Synthesis and evaluation of promising novel anticancer

compounds remains an important challenge for drug discovery

[39]. Recently, we have synthesized and characterized a series of

Levamisole derivatives and identified 4a as the most potent

molecule [21]. In the present study, we found that 4a treatment

resulted in efficient ROS production, which is an indicator of

DNA damage. Further, we show that 4a induces cytotoxicity by

activating the extrinsic pathway of apoptosis.

EAC cells possessing malignant features of cancer are used

commonly for inducing tumors in Swiss albino mice, and for

evaluating anti-cancer activity of small molecules in vivo [26–

28,40,41]. Our results show that 4a treatment led to a significant

reduction in tumor size. More than 4-fold increase in lifespan of

treated mice was observed after 4a treatment, when compared

after 48 h of treatment with 4a (5 mM), DMSO treated cells were used as control (C), western blotting was performed using anti-CYTOCHROME C.Actin was used as loading control.doi:10.1371/journal.pone.0043632.g004

Figure 5. Comparison of effect of 4a and Levamisole on progression of solid tumor in mice. Solid tumor was induced in Swiss albino miceby injecting EAC cells. Six doses of 4a and Levamisole (20 mg/kg) each administered to tumor bearing mice on every alternate day from 12th day ofEAC cell injection. A. Effect of 4a and Levamisole on tumor progression at different time points. Data shown is based on two independent batches ofexperiments containing four animals each. Error bars indicate SD from independent experiments. B. Kaplan–Meier survival curves of mice treatedwith 4a. Out of 24 tumor induced Swiss Albino animals, 12 were treated with 4a (20 mg/kg) and survival graph was plotted, Log-rank statistical testshowed P,0.005 (**). In control case, median survival time was found to be 59 days and in case of 4a treated it is undefined (value showed up to 250days). C. Gross appearance of 4a treated and untreated tumor mice and their selected organs at 25th day of treatment. a. mouse with no tumor, b.mouse bearing tumor, c. tumor bearing mouse after treatment with 4a, d. thigh tissue of normal mouse, e. tumor, f. thigh tissue of a treated mouse,g. liver from normal mouse, h. liver of a tumor mouse, i. liver from a 4a treated mouse, j. spleen of a normal mouse, k. spleen of a mouse with tumor,l. spleen of a treated mouse.doi:10.1371/journal.pone.0043632.g005

Levamisole Derivative: Novel Anti-Cancer Agent

PLOS ONE | www.plosone.org 8 September 2012 | Volume 7 | Issue 9 | e43632

Figure 6. Evaluation of side effects of Levamisole and 4a in Swiss Albino mice. 4a or Levamisole were orally administered (20 mg/kg, sixdoses in interval of two weeks) to experimental animals and body weight was monitored on 20th or 50th day, blood was collected and serum waschecked for alkaline phosphatase (ALP), creatinine; urea, and plasma was used for counting RBCs and WBCs to analyze the side effects. A, C.Evaluation of kidney and liver function following 20 and 50 days, respectively, of 4a treatment. B, D. Assessment of body weight changes in micefollowing 20 and 50 days after 4a and Levamisole treatment. Value of serum tests and blood counts are given with mean6SEM (n = 6), average bodyweight of each group was plotted with standard error.doi:10.1371/journal.pone.0043632.g006

Figure 7. Immunostaining studies for apoptotic and DNA damage markers following treatment with 4a. A–F. Ki67, BID and 53BP1immunostaining of tumor and treated tissues. The images were quantified using ImageJ software and standard error was plotted using independentimages. A, B. Antibody staining for Ki67 on 25th day tumor tissue (a, b) and tumor tissues treated with 4a (c, d) and their quantification. C, D.Immunostaining for BID on 25th day control tumor (a, b) and 4a treated tumor (c,d) and their quantification. E, F. 53BP1 staining on 25th day tumortissue (a, b) and 4a treated tumor tissue (c, d) and their quantification. Magnification of images shown in panels a and c are 106, while b and d are206.doi:10.1371/journal.pone.0043632.g007

Levamisole Derivative: Novel Anti-Cancer Agent

PLOS ONE | www.plosone.org 9 September 2012 | Volume 7 | Issue 9 | e43632

with untreated animals with tumor. Histological evaluation of

tumor and normal tissues following compound treatment further

indicates that its effect was mostly restricted to tumor cells. Thus,

effectiveness of 4a at low concentrations in mice makes it a

potential cancer therapeutic agent.

Interestingly, Levamisole, the parental compound failed to show

any cytotoxic or antitumor activity at concentrations equivalent to

4a. There are contradicting reports on anticancer activity of

Levamisole in the literature. In one of the studies, Levamisole

failed to show any anticancer activity even at higher concentra-

tions [42]. Howerever, other studies have reported that Levam-

isole can act as a potent anticancer drug in EAC as well as other

cancer cell lines [18,43,44]. It has also been shown that

Levamisole can act as immunomodulatory agent. Interestingly, it

could enhance the effect of anticancer drugs such as chlorambucil,

when used together, by acting as an immunostimulator [43].

Although combined therapy of Levamisole along with other

anticancer agents increases sensitivity of Ehrlich ascites carcinoma,

it has been demonstrated to have adverse effects on liver and

kidney metabolism and pathology. In the present study also, we

noticed hepatic abnormalities in case of Levamisole. On the other

hand, 4a, despite being a more potent anticancer compound had

limited adverse effect on histopathology or metabolic functions of

liver and kidney.

Immunohistochemical studies showed regression of tumor cell

proliferation as evident by Ki67 stained cells following 4atreatment, which was also consistent in case of western blot

analysis, where we observed downregulation of PCNA after

Figure 8. Comparison of expression of apoptotic proteins in 4a treated solid and liquid tumors in mice. A. 4a was orally administeredto mice bearing solid tumor (6 doses, 20 mg/kg). Tumor tissues were collected after 25 days of 4a treatment; lysate was prepared and used forwestern blotting. B. Expression of apoptotic proteins following 4a treatment in liquid tumor. EAC cells were injected intraperitoneally in mice togenerate liquid tumor. Following 4a treatment (6 doses, 20 mg/kg) tumor cells were collected, lysate was prepared and used for western blotting.Antibodies used were BCL2, BAD, BAX, Phospho p53, p53, PCNA, CYTOCHROME C, FAS, FAS-L, FADD, CASPASE-8 and CASPASE-3. Actin was used asloading control (A, B).doi:10.1371/journal.pone.0043632.g008

Levamisole Derivative: Novel Anti-Cancer Agent

PLOS ONE | www.plosone.org 10 September 2012 | Volume 7 | Issue 9 | e43632

treatment with 4a in tumor lysate. Elevated expression of

proapoptotic protein BID and damage sensor 53BP1, were also

observed in tumor treated tissues, suggesting the activation of

apoptosis following 4a treatment. These results suggest that 4atreatment significantly inhibits tumor cell proliferation and

increase the life span of 4a treated mice.

p53 is one of the most well studied transcription factors that

plays a critical role in cell cycle arrest, apoptosis and DNA repair

in response to a variety of cellular stresses, including DNA damage

[45,46]. 4a treatment resulted in a dose-dependent upregulation

of p53, which could be a result of ROS-mediated disruption of

mitochondrial membrane potential and DNA damage. p53

mediated transcriptional activation could regulate activation of

pro-apoptotic protein BAX [47] which in turn changes the

mitochondrial membrane potential resulting in the release of

CYTOCHROME C [48,49]. Based on our results, it is evident

that overproduction of intracellular ROS, upregulation of p53 and

release of CYTOCHROME C into cytosol, would result in the

p53 mediated apoptosis. Further, p53 upregulation can modulate

the expression of PUMA, a BCL2 family protein and an important

mediator of p53-dependent apoptosis [50,51]. Consistent with that

we found an upregulation of PUMA, upon treatment with 4a(Fig. 4A). Recently, a study showed necrotic mode of cell death by

p53 under oxidative stress, independent of caspase cleavage. This

study also showed release of CYTOCHROME C into the cytosol

upon addition of p53 to purified mitochondria [52]. Although, 4acould induce ROS production at early time points, its levels were

not constant or maintained, and this transient ROS production

did not result in necrosis. Instead, it led to phosphorylation of p53,

cleavage of CASPASE-8 and CASPASE-3, further culminating in

the activation of apoptosis.

Although, the level of cell survival protein, AKT and its

phosphorylated form p-AKT, increased after treatment with 4a, it

failed to show any effect on survival of the cell. As described above,

it is possible that upregulation of p53 and its phosphorylated form

may be sufficient to overcome the effect due to AKT.

Consistent with the above conclusion, we observed that K562

cells were much less sensitive to 4a with an IC50 value of 70, unlike

the other three leukemic cell lines studied. Ours and other groups

have shown that K562 does not express wild type p53 [53–55].

This suggests that in the absence of p53, 4a is unable to induce a

comparable level of apoptosis suggesting that it might act in a p53

dependent manner. However, this needs to be investigated further.

Generally during apoptosis, increase in proapoptotic proteins

and decrease in the levels of antiapoptotic proteins are required for

maintaining the ratio between them. However, upon addition of

4a, we observed an interesting upregulation of antiapoptotic

proteins leading to imbalance in the overall ratio and finally

resulting into apoptosis. Previous studies have also reported an

upregulation of BCL2 followed by activation of apoptosis [56,57].

In the present study, we observed a dose dependent upregula-

tion of FAS after 4a treatment in both cell lines and mouse tumor

models (Fig. 8). Induction of apoptosis through cell surface death

receptors (FAS and FAS-L) results in the activation of an initiator,

CASPASE-8. Activation of death receptors with their ligands

provokes the recruitment of adaptor proteins, such as the FAS-

associated death domain proteins (FADD), which in turn recruit

and aggregate CASPASE-8, thereby promoting its auto processing

and activation (Fig. 4D). Activated CASPASE-8 proteolytically

processes and activates CASPASE-3 that culminates in substrate

proteolysis leading to cell death. Consistent to this, we observed an

upregulation of the death receptor proteins, FAS and FAS-L in

CEM cells. Our results suggest that CASPASE-8 and CASPASE-3

Figure 9. Proposed model for mechanism of 4a induced cytotoxicity by induction of apoptosis. 4a treatment resulted in production ofROS, thereby damaging the DNA, which in turn helped in upregulation and phosphorylation of p53, where it activated extrinsic pathway of apoptosisby activating FAS, cleavage of FAS-L. These activated death receptors resulted in the recruitment of adaptor proteins, FAS-associated death domainproteins (FADD), which recruits and aggregates CASPASE-8, thereby promoting its auto processing and activation. Activated CASPASE-8 cleaves BIDinto t-BID, which further facilitates in the release of CYTOCHROME C from mitochondria, further cleaving PROCASPASE-3 into the effector CASPASE-3which leads to cell death.doi:10.1371/journal.pone.0043632.g009

Levamisole Derivative: Novel Anti-Cancer Agent

PLOS ONE | www.plosone.org 11 September 2012 | Volume 7 | Issue 9 | e43632

cleavage in 4a treated CEM cells could result in DNA

fragmentation and apoptosis (Fig. 4D).Moreover, we observed

cleavage of BID by CASPASE-8 into its truncated version t-BID

which in turn facilitates the mitochondrial pathway of apoptosis

(Fig. 4B). The mitochondrial protein, SMAC/DIABLO, plays an

important role in apoptosis by eliminating the inhibitory effect of

IAPs (inhibitor of apoptosis proteins) on caspases [58]. Our results

show a dose dependent activation of SMAC/DIABLO upon

treatment with 4a.

In summary, 4a treatment resulted in an increase in DNA

damage which led to the upregulation of p53. 4a treatment

activates FAS and FAS-L death receptor pathway, leading to

cleavage of CASPASE-8 followed by activation of CASPASE-3

(Fig. 9). Thus, the extrinsic pathway of apoptosis is induced by 4aleading to cell death both in vivo and ex vivo suggesting that 4acould be used as a potential cancer therapeutic agent.

Supporting Information

Figure S1 Lactate dehydrogenase release assay on 4a treated

CEM cells at different timepoints to evaluate the cell damage

caused by 4a.

(PPT)

Figure S2 Determination of ROS production following 4atreatment on CEM cells at different timepoints.

(PPT)

Figure S3 Histological sections of thigh and liver tissues of 4atreated and untreated mice.

(PPT)

Figure S4 Histological sections of liver and brain tissues of from

4a or Levamisole treated normal mice.

(PPT)

Acknowledgments

We thank Dr. Mridula Nambiar, Ms. Sheetal Sharma, Ms. Nishana M.,

Ms. Mrinal Srivastava and members of the SCR laboratory for discussions

and help. We would also like to thank NMR facility, Indian Institute of

Science, Bangalore for characterization of 4a.

Author Contributions

Conceived and designed the experiments: SCR MH SSK. Performed the

experiments: MH SSK ET SK KP SRR KSR BC. Analyzed the data:

SCR MH BC. Wrote the paper: SCR MH BC.

References

1. Nambiar M, Kari V, Raghavan SC (2008) Chromosomal translocations in

cancer. Biochim Biophys Acta 1786: 139–152.

2. Nickoloff JA, De Haro LP, Wray J, Hromas R (2008) Mechanisms of leukemia

translocations. Curr Opin Hematol 15: 338–345.

3. Aplan PD (2005) ‘‘You break it, you fix it.’’. Blood 105: 1843–1844.

4. Rowley JD (2001) Chromosome translocations: dangerous liaisons revisited. Nat

Rev Cancer 1: 245–250.

5. Nambiar M, Raghavan SC (2011) How does DNA break during chromosomal

translocations? Nucleic Acids Res 39: 5813–5825.

6. Raghavan SC, Swanson PC, Wu X, Hsieh CL, Lieber MR (2004) A non-B-

DNA structure at the Bcl-2 major breakpoint region is cleaved by the RAG

complex. Nature 428: 88–93.

7. Kumar-Sinha C, Tomlins SA, Chinnaiyan AM (2008) Recurrent gene fusions in

prostate cancer. Nat Rev Cancer 8: 497–511.

8. Nussenzweig A, Nussenzweig MC (2010) Origin of chromosomal translocations

in lymphoid cancer. Cell 141: 27–38.

9. Moen MD, McKeage K, Plosker GL, Siddiqui MA (2007) Imatinib: a review of

its use in chronic myeloid leukaemia. Drugs 67: 299–320.

10. Robert J, Jarry C (2003) Multidrug resistance reversal agents. J Med Chem 46:

4805–4817.

11. Huang C, Yang S, Ge R, Sun H, Shen F, et al. (2008) ZNF23 induces apoptosis

in human ovarian cancer cells. Cancer Lett 266: 135–143.

12. Tang DG, Porter AT (1997) Target to apoptosis: a hopeful weapon for prostate

cancer. Prostate 32: 284–293.

13. Buolamwini JK (2000) Cell cycle molecular targets in novel anticancer drug

discovery. Curr Pharm Des 6: 379–392.

14. Stevenson HC, Green I, Hamilton JM, Calabro BA, Parkinson DR (1991)

Levamisole: known effects on the immune system, clinical results, and future

applications to the treatment of cancer. J Clin Oncol 9: 2052–2066.

15. Kovach JS, Svingen PA, Schaid DJ (1992) Levamisole potentiation of

fluorouracil antiproliferative activity mimicked by orthovanadate, an inhibitor

of tyrosine phosphatase. J Natl Cancer Inst 84: 515–519.

16. Friis T, Engel AM, Klein BM, Rygaard J, Houen G (2005) Levamisole inhibits

angiogenesis in vitro and tumor growth in vivo. Angiogenesis 8: 25–34.

17. Guminska M, Kedryna T, Marchut E (1986) The effect of levamisole on energy

metabolism in Ehrlich ascites tumour cells in vitro. Biochem Pharmacol 35:

4369–4374.

18. Artwohl M, Holzenbein T, Wagner L, Freudenthaler A, Waldhausl W, et al.

(2000) Levamisole induced apoptosis in cultured vascular endothelial cells.

Br J Pharmacol 131: 1577–1583.

19. Andreani A, Bonazzi D, Rambaldi M (1982) Heterocyclisierungen unter

Verwendung cyclischer Hydrazinium- und Hydrazoniumdithiokohlensauredie-

ster-Salze zu zwei- und dreikernigen S,N-Heterocyclen. Archiv der Pharmazie

315: 451–456.

20. Gadad AK, Karki SS, Rajurkar VG, Bhongade BA (1999) Synthesis and

biological evaluation of 5-formyl-6-arylimidazo(2,1-b)-1,3,4-thiadiazole-2-N-

(dimethylaminomethi no) sulfonamides as antitumor agents. Arzneimittel-

forschung 49: 858–863.

21. Karki SS, Panjamurthy K, Kumar S, Nambiar M, Ramareddy SA, et al. (2011)

Synthesis and biological evaluation of novel 2-aralkyl-5-substituted-6-(49-

fluorophenyl)-imidazo[2,1-b][1,3,4]thiadiazo le derivatives as potent anticancer

agents. Eur J Med Chem 46: 2109–2116.

22. Shahabuddin MS, Nambiar M, Moorthy BT, Naik PL, Choudhary B, et al.

(2011) A novel structural derivative of natural alkaloid ellipticine, MDPSQ,

induces necrosis in leukemic cells. Invest New Drugs 29: 523–533.

23. Chiruvella KK, Kari V, Choudhary B, Nambiar M, Ghanta RG, et al. (2008)

Methyl angolensate, a natural tetranortriterpenoid induces intrinsic apoptotic

pathway in leukemic cells. FEBS Lett 582: 4066–4076.

24. Korzeniewski C, Callewaert DM (1983) An enzyme-release assay for natural

cytotoxicity. J Immunol Methods 64: 313–320.

25. Chiruvella KK, Raghavan SC (2011) A natural compound, methyl angolensate,

induces mitochondrial pathway of apoptosis in Daudi cells. Invest New Drugs 4:

583–592.

26. Noaman E, Badr El-Din NK, Bibars MA, Abou Mossallam AA, Ghoneum M

(2008) Antioxidant potential by arabinoxylan rice bran, MGN-3/biobran,

represents a mechanism for its oncostatic effect against murine solid Ehrlich

carcinoma. Cancer Lett 268: 348–359.

27. Ghosh M, Bhattacharya S, Sadhu U, Dutta S, Sanyal U (1997) Evaluation of

beta-tethymustine, a new anticancer compound, in murine tumour models.

Cancer Lett 119: 7–12.

28. Attia MA, Weiss DW (1966) Immunology of spontaneous mammary carcinomas

in mice. V. Acquired tumor resistance and enhancement in strain A mice

infected with mammary tumor virus. Cancer Res 26: 1787–1800.

29. Sharma S, Panjamurthy K, Choudhary B, Srivastava M, Shahabuddin M, et al.

(2012) A novel DNA intercalator, 8-methoxy pyrimido[49,59:4,5]thieno (2,3-

b)quinoline-4(3H)-one induces apoptosis in cancer cells, inhibits the tumor

progression and enhances lifespan in mice with tumor. Mol Carcinog.

30. Bhattacharyya A, Choudhuri T, Pal S, Chattopadhyay S, Datta KG, et al.

(2003) Apoptogenic effects of black tea on Ehrlich’s ascites carcinoma cell.

Carcinogenesis 24: 75–80.

31. Hazra B, Kumar B, Biswas S, Pandey BN, Mishra KP (2005) Enhancement of

the tumour inhibitory activity, in vivo, of diospyrin, a plant-derived quinonoid,

through liposomal encapsulation. Toxicol Lett 157: 109–117.

32. Kumagai H, Mukaisho K, Sugihara H, Miwa K, Yamamoto G, et al. (2004)

Thioproline inhibits development of esophageal adenocarcinoma induced by

gastroduodenal reflux in rats. Carcinogenesis 25: 723–727.

33. Vrekoussis T, Chaniotis V, Navrozoglou I, Dousias V, Pavlakis K, et al. (2009)

Image analysis of breast cancer immunohistochemistry-stained sections using

ImageJ: an RGB-based model. Anticancer Res 29: 4995–4998.

34. Luo X, Budihardjo I, Zou H, Slaughter C, Wang X (1998) Bid, a Bcl2

interacting protein, mediates cytochrome c release from mitochondria in

response to activation of cell surface death receptors. Cell 94: 481–490.

35. Luppi G, Zoboli A, Barbieri F, Crisi G, Piccinini L, et al. (1996) Multifocal

leukoencephalopathy associated with 5-fluorouracil and levamisole adjuvant

therapy for colon cancer. A report of two cases and review of the literature. The

INTACC. Intergruppo Nazionale Terpia Adiuvante Colon Carcinoma. Ann

Oncol 7: 412–415.

36. Symoens J, Veys E, Mielants M, Pinals R (1978) Adverse reactions to levamisole.

Cancer Treat Rep 62: 1721–1730.

Levamisole Derivative: Novel Anti-Cancer Agent

PLOS ONE | www.plosone.org 12 September 2012 | Volume 7 | Issue 9 | e43632

37. Gerdes J, Lemke H, Baisch H, Wacker HH, Schwab U, et al. (1984) Cell cycle

analysis of a cell proliferation-associated human nuclear antigen defined by the

monoclonal antibody Ki-67. J Immunol 133: 1710–1715.

38. Cheung AN, Ngan HY, Chen WZ, Loke SL, Collins RJ (1993) The significance

of proliferating cell nuclear antigen in human trophoblastic disease: an

immunohistochemical study. Histopathology 22: 565–568.

39. Sugimoto Y, Konoki K, Murata M, Matsushita M, Kanazawa H, et al. (2009)

Design, synthesis, and biological evaluation of fluorinated analogues of

salicylihalamide. J Med Chem 52: 798–806.

40. Kumar A, D’Souza SS, Nagaraj SR, Gaonkar SL, Salimath BP, et al. (2009)

Antiangiogenic and antiproliferative effects of substituted-1,3,4-oxadiazole

derivatives is mediated by down regulation of VEGF and inhibition of

translocation of HIF-1alpha in Ehrlich ascites tumor cells. Cancer Chemother

Pharmacol 64: 1221–1233.

41. Gekeler V, Epple J, Kleymann G, Probst H (1993) Selective and synchronous

activation of early-S-phase replicons of Ehrlich ascites cells. Mol Cell Biol 13:

5020–5033.

42. Wiebke EA, Grieshop NA, Loehrer PJ, Eckert GJ, Sidner RA (2003) Antitumor

effects of 5-fluorouracil on human colon cancer cell lines: antagonism by

levamisole. J Surg Res 111: 63–69.

43. Salem FS, Badr MO, Neamat-Allah AN (2011) Biochemical and pathological

studies on the effects of levamisole and chlorambucil on Ehrlich ascites

carcinoma-bearing mice. Vet Ital 47: 89–95.

44. Ramanadham M, Nageshwari B (2010) Anti-proliferative effect of levamisole on

human myeloma cell lines in vitro. J Immunotoxicol 7: 327–332.

45. Levine AJ (1997) p53, the cellular gatekeeper for growth and division. Cell 88:

323–331.

46. Agarwal ML, Taylor WR, Chernov MV, Chernova OB, Stark GR (1998) The

p53 network. J Biol Chem 273: 1–4.

47. Miyashita T, Reed JC (1995) Tumor suppressor p53 is a direct transcriptional

activator of the human bax gene. Cell 80: 293–299.48. Jurgensmeier JM, Xie Z, Deveraux Q, Ellerby L, Bredesen D, et al. (1998) Bax

directly induces release of cytochrome c from isolated mitochondria. Proc Natl

Acad Sci U S A 95: 4997–5002.49. Rosse T, Olivier R, Monney L, Rager M, Conus S, et al. (1998) Bcl-2 prolongs

cell survival after Bax-induced release of cytochrome c. Nature 391: 496–499.50. Yu J, Zhang L (2008) PUMA, a potent killer with or without p53. Oncogene 27

Suppl 1: S71–83.

51. Li PF, Dietz R, von Harsdorf R (1999) p53 regulates mitochondrial membranepotential through reactive oxygen species and induces cytochrome c-indepen-

dent apoptosis blocked by Bcl-2. EMBO J 18: 6027–6036.52. Vaseva AV, Marchenko ND, Ji K, Tsirka SE, Holzmann S, et al. (2012) p53

Opens the Mitochondrial Permeability Transition Pore to Trigger Necrosis. Cell149: 1536–1548.

53. Sallmyr A, Tomkinson AE, Rassool FV (2008) Up-regulation of WRN and DNA

ligase IIIalpha in chronic myeloid leukemia: consequences for the repair of DNAdouble-strand breaks. Blood 112: 1413–1423.

54. Kumar TS, Kari V, Choudhary B, Nambiar M, Akila TS, et al. (2010) Anti-apoptotic protein BCL2 down-regulates DNA end joining in cancer cells. J Biol

Chem 285: 32657–32670.

55. Durland-Busbice S, Reisman D (2002) Lack of p53 expression in human myeloidleukemias is not due to mutations in transcriptional regulatory regions of the

gene. Leukemia 16: 2165–2167.56. Knudson CM, Korsmeyer SJ (1997) Bcl-2 and Bax function independently to

regulate cell death. Nat Genet 16: 358–363.57. Tsujimoto Y (1998) Role of Bcl-2 family proteins in apoptosis: apoptosomes or

mitochondria? Genes Cells 3: 697–707.

58. Chai J, Du C, Wu JW, Kyin S, Wang X, et al. (2000) Structural and biochemicalbasis of apoptotic activation by Smac/DIABLO. Nature 406: 855–862.

Levamisole Derivative: Novel Anti-Cancer Agent

PLOS ONE | www.plosone.org 13 September 2012 | Volume 7 | Issue 9 | e43632